Calcium influx via dihydropyridine-sensitive, voltage-gated L-type calcium channels (LTCC) plays a crucial role in the regulation of excitability, contraction, and gene expression in arterial smooth muscle. Exaggerated Ca2+ influx through smooth muscle LTCCs has been implicated in the chain of events contributing to hyperglycemia-induced vascular dysfunction during non-insulin dependent diabetes mellitus (NIDDM). However, the molecular mechanisms underlying the increase in LTCC activity during hyperglycemia and NIDDM remain poorly defined. Recently, we identified and characterized a novel modality of LTCC function in which a single or a small cluster of these channels can operate in a persistent gating mode that create sites of nearly continual Ca2+ influx (called "persistent Ca2+ sparklets") in arterial smooth muscle. Under physiological conditions persistent Ca2+ sparklet activity is low. However, preliminary data presented in this application suggest that Ca2+ sparklet activity increases during hyperglycemia through a mechanism requiring protein kinase A (PKA) activation and targeting via scaffolding AKAP150. The goal of this application is to test the central hypothesis that an increase in persistent Ca2+ sparklet activity is an early, critical event in the pathway leading to vascular dysfunction during diabetes. The central hypothesis has been formulated on the basis of strong preliminary data and will be tested by pursuing three novel specific aims. Aim 1 will investigate the mechanisms by which acute hyperglycemia and diabetes increases Ca2+ sparklet activity in arterial smooth muscle. Aim 2 will determine whether AKAP150 and PKA activity are required for increased Ca2+ sparklet activity during acute hyperglycemia and diabetes. Aim 3 will test the hypothesis that persistent Ca2+ sparklets downregulate K+ channel expression through the activation of NFATc3 during acute hyperglycemia and diabetes. These hypotheses will be tested using a series of novel imaging approaches developed by our team in combination with state-of-the-art electrophysiological, cellular, and molecular biological approaches. The experiments outlined in this application are significant because they will provide new fundamental information on the molecular mechanisms underlying vascular dysfunction during NIDDM and may contribute to the development of rational therapies for the treatment of this pathological condition. PUBLIC HEALTH RELEVANCE Approximately 10 million Americans suffer of non-insulin dependent diabetes and hypertension, which, if untreated, leads to several cardiovascular complications. This research project will determine the mechanisms by which hyperglycemia induces the activation of a novel Ca2+ signaling modality (e.g. Ca2+ sparklets) in the muscle cells of blood vessels, causing increased contraction and thereby leading to arterial dysfunction during diabetes and hypertension.